Easy synthesis of photoluminescent N-doped carbon dots from winter melon for bio-imaging

Abstract

N-doped carbon dots were successfully synthesized via a one-step hydrothermal method by using edible winter melon as the source material. Mono-dispersed CDs 4.5–5.2 nm in diameter were achieved in a quantum yield (QY) of 7.51%. The photoluminescent CDs were demonstrated to be effective bio-imaging agents for hepG2 (liver hepatocellular carcinoma) cells.

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Hepatocellular carcinoma is a serious malignancy and a severe threat to human health worldwide. Therefore, it is particularly significant to comprehensively elucidate the pathogenesis of hepatocellular carcinoma for diagnosis and treatment. As the intrinsic fluorescence of biological molecules is weak and non-specific, fluorescent labelling and imaging techniques have become a sensible visual analysis tool to clarify the biological process on the cellular and molecular level.¹ Traditionally, owing to the prominent advantages of well-defined structures, feasibility of synthesis, high quantum yield, and remarkable photostability,^2,3 semiconductor quantum dots (QDs), such as CdS and CdSe, have been the preferred choice for cell imaging.⁴ Unfortunately, semiconductor QDs are toxic and have poor dispersity in water, thus bringing in serious safety and health concerns, especially for the long-term imaging of cells. In addition, Pluronic F127 or hydrophilic chemicals were introduced to minimize, but not totally eliminate the toxicity of QDs.^5,6 The greatest challenge, therefore, is to confidently report the bio-imaging of the observed living cells by using non-toxic fluorescent alternatives while maintaining advantageous optical performances.

As an emerging class of carbon nanomaterials, carbon dots (CDs) exhibit strong fluorescence, outstanding photostability, biocompatibility, aqueous dispersibility and non-toxicity with favorable size advantage (below 10 nm).^7–10 These features make CDs promising candidates to replace the traditional fluorescent materials in many applications such as sensors, optoelectronic devices, bio-imaging, drug delivery and catalysis.^11–18 Many complex synthetic methods, such as laser ablation, hydrothermal oxidation, electrochemical oxidation, high-temperature calcination, solvothermal and microwave assisted pyrolysis, have been usedto prepare CDs to date.^19–25 However, two issues that need to be addressed for the effective synthesis of cost-effective CDs still remain: the size non-uniformity and the time-consuming fabrication process.^26,27 CDs have commonly been prepared by using graphite powder and rods,^19,20,28 carbon nanotubes,²⁹ carbon fibers,³⁰ carbohydrates,^21,31 ammonium citrate,³² and petroleum coke³³ as carbon sources. Recently, the utilization of eco-friendly materials has drawn much attention.³⁴ A diverse set of cheap, natural and non-toxic available materials like food waste, hair waste, potato, chitosan, cow milk, and coffee grounds have been used for the preparation of photoluminescent CDs.^24,35–42

Conventionally, the fluorescence properties and bio-imaging efficiencies of CDs are influenced by using surface passivation, which requires both passivating agents and multiple time consuming steps.^10,11 Doping CDs with other non-metallic components is beneficial for adjusting the structure and composition of CDs.⁴³ N-doped CDs have been found to efficiently induce charge delocalization and enhance performance in bio-imaging and catalysis with N atoms as dopants.⁴⁴ Winter melon is an edible and widely grown vegetable that is rich in vitamins, proteins, and carbohydrates. In this study, we utilized winter melon as both a carbon source and a nitrogen source to directly prepare photoluminescent N-doped CDs 4.5–5.2 nm in size with a quantum yield (QY) of 7.51% through efficient one-step hydrothermal treatment without rigid reaction conditions, surface passivation, or tedious post-processing. The N-doped CDs were used as fluorescent agents for liver hepatocellular carcinoma (hepG2) cell imaging.

The synthetic procedure is illustrated in Scheme 1 and more details can be seen from the ESI.† The WMJ was extracted from crushed winter melon and filtered with a 0.22 μm microporous membrane. Homogeneous and water-soluble CDs can be directly prepared by a hydrothermal process at 180 °C for 2 h after subsequent centrifugation and dialysis. The dispersibility of CDs is ascribed to the abundant functional groups derived from the carbonization of WMJ. The morphology and particle size of CDs were examined by TEM and DLS analysis, as shown in Fig. 1. Fig. 1A reveals that the N-doped CDs are uniform and regularly spherical in shape with an average diameter of about 5 nm without aggregation. The corresponding particle size measured by DLS (Fig. 1B) indicates that N-doped CDs have a relatively narrow size distribution between 4.5 and 5.2 nm. The inset in Fig. 1A is a typical HRTEM image of N-doped CDs, indicating a certain crystallinity with a lattice space of approximately 0.31 nm, which is in agreement with values in the literature.¹⁷

Scheme 1 The formation process of N-doped carbon dots by one-step hydrothermal method.

Fig. 1 (A) HRTEM image of N-doped CDs, the inset describes the magnification of a single CD; (B) particle size distribution of CDs.

XPS and FTIR measurements were performed to identify the effective incorporation of nitrogen and surface functional groups of CDs. From a survey scan of the XPS spectrum (Fig. 2A), three distinct peaks centred at 284.5 eV, 399.1 eV, and 531.1 eV can be observed, corresponding to C1s, N1s, and O1s, respectively. In detail, the C1s spectrum (Fig. 2B) displays three distinct peaks at 284.6 eV, 286.0 eV, and 287.5 eV, which are attributed to C–C, C–N, C=O, respectively.^45,46 The N1s spectrum has two typical peaks at 398.3 (pyridinic Ns) and 400.2 eV (pyrrolic Ns) in Fig. 2C,^46,47 indicating that nitrogen is present in a π-conjugated system where two p-electrons are present in the system of as-prepared N-doped CDs.⁴⁴ The FTIR spectrum in Fig. 2D shows the characteristic absorption bands at 3416 cm⁻¹ corresponding to the stretching vibrations of O–H and N–H.⁴⁸ The peak at 2926 cm⁻¹ is ascribed to the C–H bonds. The absorption bands at 1624 cm⁻¹ and 1404 cm⁻¹ are due to the C–O stretching vibrations and C–N stretching vibrations, respectively. The existence of carboxylic groups can be clearly proven by the peak at 1732 cm⁻¹ corresponding to C=O and the peak at 1071 cm⁻¹ attributed to C–O, suggesting the partial oxidation of CD surfaces.⁴⁹ Furthermore, the band centered at 1245 cm⁻¹ is assigned to the asymmetric stretching vibrations of the C–O–C bond.⁵⁰ It can be concluded from the results of XPS and FTIR analysis that the hydrothermal degradation of WMJ offered the as-synthesized N-doped CDs with hydrophilic groups such as –COOH and –OH, which are beneficial for the improvement of aqueous solubility of CDs for potential application in biochemistry, drug delivery and diagnostics.

Fig. 2 XPS and FTIR spectra of N-doped CDs. (A) XPS full scan spectrum; (B) C1s spectrum; (C) N1s spectrum; (D) FTIR spectrum.

Photoluminescence (PL) is a more attractive property in N-doped CDs than other carbon-materials. The UV-vis absorption spectra of the WMJ and N-doped CDs are given in Fig. 3. No obvious absorption band can be detected in the UV-vis spectrum of WMJ. In comparison with WMJ, N-doped CDs exhibit a strong absorption peak at around 280 nm, which can be attributed to the presence of π–π* interactions in N-doped CDs.^51,52 In addition, a broad shoulder at around 325 nm has also been found, perhaps attributable to the excited defect surface states induced by N atoms.⁴¹

Fig. 3 UV-vis absorption spectra of winter melon juice (WMJ) and N-doped CDs aqueous solutions.

Fig. 4A shows the excitation and emission spectra peaks of the N-doped CDs in aqueous solution. When irradiated under a short-wavelength laser, N-doped CDs generate strong photoluminescence between 450 and 550 nm. The N-doped CD aqueous solution is transparent and yellow in bright field and acquires a strong blue-fluorescence under UV excitation (inset, Fig. 4A). As shown in Fig. 4B, the N-doped CD solution has an obvious excitation-dependent PL behaviour. The solution presents strongest luminescence intensity when excited at 360 nm, while the maximum emission peaks are located at 448 nm. Besides, with the increase in excitation wavelength from 360 nm to 450 nm, the intensity of the emission peaks remarkably decreased and the spectra were red-shifted from 448 nm to 480 nm. The full width at half maximum (FWHM) under 360 nm excitation was calculated and the result was 80 nm, further verifying the narrow size distribution of the as-synthesized N-doped CDs. The PL stability of N-doped CDs to the effects of the pH of the solution and UV exposure temperature were investigated. The PL intensity is largely controlled by pH values and the maximum intensity is at neutral pH. The PL intensity increases significantly as the pH increases from 4 to 6, and decreases slightly in alkaline solution with the pH increasing from 8 to 13 (Fig. S1†). This stimulus-response property is advantageous for the exploitation of CD-based pH sensing systems. Furthermore, a temperature-independent PL behaviour was also observed; it can be shown that the PL intensity of the N-doped CDs is nearly unchanged at different exposure temperatures (Fig. S2†). Additionally, N-doped CDs showed excellent photostability, as the PL intensity exhibited no meaningful reduction (∼6.7%) even after continuous excitation with a Xe lamp from 0.5 days to 12 days (Fig. S3†). The QY of N-doped CDs without surface passivation in aqueous solution at pH 7 as measured at an excitation wavelength of 360 nm is estimated to be 7.51% by using quinine sulphate as standard. Thus, the excellent PL of N-doped CDs will doubtless enhance their applicability in cell imaging fields.

Fig. 4 (A) Fluorescence excitation and emission spectra of the N-doped CDs aqueous solutions, inset: photographs of the N-doped CDs aqueous solutions when exposed to daylight and 360 nm UV irradiation, respectively; (B) emission spectra of the N-doped CDs at different excitation ranged from 340 to 450 nm with a 10 nm increase in each step.

In order to investigate the biocompatibility of the as-synthesized N-doped CDs, a CKK-8 assay was used to assess the cytotoxicity of N-doped CDs by using hepG2 cells, and the results are shown in Fig. 5. Cell viability was barely affected when N-doped CD concentration varied from 0 to 0.8 mg mL⁻¹ for 24 h. The cell survival rates still exceeded 90% at all the experimental concentrations, indicating that the N-doped CDs have no apparent toxicity and are cyto-compatible with hepG2 cells, which makes them a potential candidate for imaging in biosensors and in vivo. Based on the excellent photoluminescence properties, aqueous dispersibility, narrow size distribution, and low cytotoxicity, N-doped CDs can serve as promising probes for cell imaging applications. Here the hepG2 cells were incubated with an N-doped CD concentration of 0.8 mg mL⁻¹ and the imaging performance was examined to assess the cell viability by confocal fluorescence microscopy. As shown in Fig. 6, all the hepG2 cells showing bright blue fluorescence under UV light excitation can be clearly imaged, and no damage was observed in the microscope after labelling with N-doped CDs. The N-doped CDs are mainly internalized into the cytoplasm region surrounding the nucleus. All the above demonstrates that N-doped CDs prepared from winter melon can be taken up by hepG2 cells and serve as a promising bio-imaging agent.

Fig. 5 CKK-8 cytotoxicity assay of hepG2 cells incubated with N-doped CDs at different concentrations for 24 h.

Fig. 6 Confocal laser microscopic image of hepG2 cells after the cellular uptake of N-doped CDs (0.8 mg mL⁻¹) for 24 h. The image was obtained by 405 nm excitation and the emission is recorded at 420–520 nm.

Conclusions

In summary, an efficient one-step hydrothermal route was successfully utilized to synthesize photoluminescent N-doped CDs from winter melon. To the best of our knowledge, this is the first time that N-doped CDs are directly prepared using winter melon juice as both a carbon source and a nitrogen source without any additional surface passivation. The obtained N-doped CDs with a narrow size distribution from 4.5 nm to 5.2 nm exhibit outstanding aqueous dispersibility, strong photoluminescence, and excellent photostability. Based on the favorable biocompatibility and low cytotoxicity as verified by CKK-8 assay, the N-doped CDs were internalized into hepG2 cells as cell-imaging agents showing bright blue fluorescence at UV light excitation. Moreover, the N-doped CDs produced from an edible and widely grown vegetable would strengthen the application prospect for green type fluorescent labels, drug delivery agents, and bio-sensors.

Acknowledgements

This work was financially supported by the Science and Technology Commission of Shanghai Municipality (13ZR1415100, 13JC1402700, 15ZR1415100). The authors are also grateful to Instrumental Analysis & Research Center of Shanghai University.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experiment section. See DOI: 10.1039/c5ra02271a

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